PROJECT SUMMARY There is a substantial need for the development of preventative and therapeutic options to delay the aging process, to rejuvenate tissue/organs, and to enhance regeneration and repair. Aging of the blood system is associated with loss of vascular integrity and dramatic changes in hematopoietic stem cell (HSC) function. In addition to increasing in number and losing self-renewal potential, old HSCs exhibit a myeloid bias and increased propensity to develop hematologic malignancies. While some of these changes reflect cell-intrinsic alterations, emerging evidence suggests that some of these defects may also be regulated by the bone marrow (BM) microenvironment, in particular the BM endothelial niche. In particular, we have demonstrated that the BM endothelial niche is indispensable for supporting the balance between HSC self-renewal and differentiation following myeloablative insult. We have also shown that AKT/mTOR signaling in BM endothelial cells (BMECs) is required for maintenance of the HSCs and that this signaling is disrupted in aged BMECs. To ask how a disruption of the AKT/mTOR signaling axis in ECs affects the hematopoietic system, we have conditionally deleted mTOR in adult ECs (mTOR(ECKO) mice) and found that this deletion resulted in the premature aging of HSCs as demonstrated by their phenotypic, functional, and transcriptional analysis. To better understand how EC-mediated signaling drives the aging of the hematopoietic system, we utilized our mTOR(ECKO) mouse model as a screening tool to discover as yet unknown pro-HSC-aging factors. Transcriptomic and proteomic studies identified Thrombospondin-1 (TSP1) as a candidate pro-aging factor that was commonly upregulated in mTOR(ECKO) and aged mice when compared to young controls. Moreover, global knockout of TSP1 resulted in preservation and rejuvenation of HSC function in aged mice. This research proposal is designed to test whether it is possible to: 1) reverse the age-related phenotypes of the hematopoietic and vascular systems by global or cell-specific deletion, or antibody-mediated inhibition, of TSP1, 2) enhance the ex vivo expansion and function of aged HSC by exogenous inhibition of TSP1, and 3) rejuvenate an aged BM microenvironment following myelosuppressive treatments by utilizing young BMECs as a cellular therapeutic with or without the co-infusion of a neutralizing antibody to TSP1. The proposed studies will utilize novel in vivo and in vitro models developed in our lab and, if successful, will unlock the therapeutic potential of TSP1 inhibition to improve overall healthspan, longevity, and regeneration of the hematopoietic system in the elderly. The success of this research proposal will open up new avenues for the development of a wide array of therapeutic strategies designed as an effective means to reverse age-related hematopoietic deficiencies.